Astaxanthin production using fed-batch fermentation process by Phaffia rhodozyma

ABSTRACT

Disclosed is a fermentation method of astaxanthin using Phaffia rhodozyma comprising the steps of: (a) in the growing phase, feeding of a nutrient medium containing glucose or sucrose based on the specific growth rate (μ) of Phaffia rhodozyma cells, and (b) in the astaxanthin production phase, feeding of the nutrient medium based on the astaxanthin production rate, while keeping the glucose concentration in the fermentation broth almost 0 g/L during the whole fermentation period.

This application is the National Stage of International Application No.PCT/EP2003/010293, filed Sep. 16, 2003.

The present invention provides a process for producing astaxanthin inhigh yield by Phaffia rhodozyma (P. rhodozyma) utilizing the fed-batchfermentation system with application of new feeding methods based on thecell growth, the carbon source consumption and the carotenoidsproduction.

Astaxanthin, as one of well-known carotenoids, is a xanthophyll commonlyfound as red or orange pigment in marine environments. Since thispigment is considered as a characteristic color in certain marineanimals that cannot synthesize β-carotene de novo such as salmon,crustaceans and trouts, it is therefore necessary to add it to theirdiet in order to give them a suitable color that appeals to theconsumer. This carotenoid is also useful for adding pigmentation to theflesh and products of other animals, and to other foodstuffs, e.g.poultry and eggs, various dairy products, snack foods, and the like.Only a few microorganisms synthesize astaxanthin, of which the yeast P.rhodozyma is a possible candidate for commercial production because ofits high astaxanthin content. P. rhodozyma is known as a carotenogenicyeast strain which produces astaxanthin specifically. Different from theother carotenogenic yeast, Rhodotorula spec., P. rhodozyma can fermentsome sugars such as D-glucose. This is an important feature from aviewpoint of industrial application. In a recent taxonomic study, asexual cycle of P. rhodozyma was revealed and its telemorphic state wasdesignated under the name of Xanthophyllomyces dendrorhous. However,natural isolates of P. rhodozyma produce so little astaxanthin(typically 100 to 300 parts per million (ppm)) that they are notpractical or economical pigment sources for aquaculture. If Phaffiastrains are to be an economically feasible feed additive for coloringaquatic animals, or any other potential food stuff (animal orotherwise), then astaxanthin over-producing strains must be developed.Mutants of naturally occurring “wild-type” Phaffia have been describedin literature, allegedly capable of generating higher levels ofastaxanthin than the wild-type yeasts. These strains reportedly producehigher levels of astaxanthin than the wild-type isolates under-specificconditions.

Besides the need to develop a suitable strain of P. rhodozyma forcommercial carotenoids production, methods for cultivating P. rhodozymaalso need to be developed which maximize carotenoids production in largefermentors.

The present invention provides a process for the production ofastaxanthin in high yield comprising applying new feeding methods ofnutrient media based on the cell growth, the carbon source consumptionand the carotenoids productions in the fed-batch mode by Phaffiastrains. Especially, by using the new feeding methods of nutrient mediabased on the carbon source consumption, the fermentation period could beshortened and the astaxanthin yield could be significantly enhanced

The present invention relates to a fermentation process for productionof astaxanthin in high yield by P. rhodozyma which applies the newfeeding methods of nutrient media based on the cell growth, the carbonsource consumption and the carotenoids productions in the fed-batchfermentation system.

It is well known that the carotenoids production by P. rhodozyma istypical non-growth associated type fermentation. It is thought thathigher cell yield against the consumed carbon source (Yx/s) is obtainedin the growth phase and lower Yx/s was obtained in the production phase.The present invention provides the new feeding strategies which comprisethe exponential feeding methods combined with the carbon sourceconcentration control and the feeding methods based on the carbonsources consumption rates in the aim of enhancing the cell growthextremely in the growth phase which possesses higher Yx/s, andregulating the cell growth and efficiently convert glucose tocarotenoids in the production phase.

The present invention relates to a process for production of astaxanthinin high yield by P. rhodozyma utilizing the fed-batch fermentationsystem. Especially, the new feeding methods based on the cell growth,the carbon source consumption and the carotenoids productions wereconstructed.

The Phaffia strain is propagated by being transferred from a slant to500 ml Erlenmeyer flasks containing 50 ml of nutrient medium in whichthe cells are cultured with shaking under sufficient aeration at 20-22°C. for 3 days. Thus this seed culture is transferred to 500 mlErlenmeyer flasks containing 100 ml of nutrient medium, and incubatedfor 2 days on a rotary shaker under sufficient aeration at 20-22° C.Aliquots of these cell cultures are then, used to inoculate into thefermentors.

The seed culture is transferred to a 5 L fermentor containing 1.75 L ofthe nutrient medium. The culture was subjected to batch growth at 20-22°C. until a yeast dry matter content of 1 g/L was obtained. Thereafter,the solution of sugar supply was started and the fed-batch fermentationwas performed at 20-22° C.

The nutrient medium basically contains glucose or sucrose as a carbonsource and nitrogen sources supplemented with various vitamins andminerals in assimilable form. Typical of these vitamins and minerals areammonium sulphate, potassium phosphate, magnesium sulphate, zincsulphate, ferric ammonium sulphate, copper sulphate, inositol,pyridoxine hydrochloride, thiamine, calcium pantothenate, biotin, andthe like. The combinations and concentrations of these materials,including the glucose and yeast extracts, can vary to convenience. Ifdesired, an antifoam agent and/or other additives can also beincorporated into or used with the medium. The medium, which was fed tothe fermentor in the fed-batch fermentation, contained a polysaccharide,e.g. glucose or sucrose.

The nutrient medium can contain polymerized forms of glucose, sucroseand other polysaccharides, molasses and corn syrup, glycerol and otherpolyols, carboxylic acids as the energy sources. Meanwhile, the nutrientmedium can contain yeast extract, meat-extract, peptone, casein, cornsteep liquor, urea, amino acidsnitrates, ammonium salts and the like asthe nutrient sources.

The fermentor with the nutrient medium is sterilized by autoclaving. Theworking volume is not restricted and the fermentation can be operatedfrom the small scale to the industrial large scale. The pH of the mediais usually maintained between 4.5 and 7.0, and the temperature between15-24° C. The medium is usually sparged with filter sterilized air, andit is continuously agitated. The strains are propagated in fermentorsover a pH range of 4.5 to 7.0 controlled with NH₄OH solution, NaOHsolution or both. Temperature can be set at a range of from 15 to 24° C.and DO is controlled by agitation and airflow to between 10% and 90%.

1. Typical feeding method: A typical feeding method for nutrient mediais disclosed in U.S. Pat. No. 6,015,684. During the growth phase, thefeeding medium containing glucose or sucrose is fed to the mainfermentor with the aim of reaching the specific growth rate of the yeastcells of μ: 0.01-0.10 h⁻¹. And the feeding rate is gradually increasedto so as not to accumulate the nutrient and ethanol in the culturebroth. After the desired yeast solids is achieved and prior to theaccumulation of the nutrient source, the feed rate is reduced to about50% of the maximum feed rate during the growth phase. During this periodof reduced nutrient source feed, namely the production phase, the yeastcells continue to produce astaxanthin, but the growth in the number ofcells is restricted. Fermentations typically last 4-9 days, and they aresampled periodically for analysis of cell growth and astaxanthinproduction.

Specific glucose consumption rate (q) and the specific growth rate (μ)can be calculated. μ is increased with q in the relationship between qand μ. The slope μ/q means the cell yield against consumed glucose(Yx/s) as the equation (1).μ/q={(1/X)·(dX/dt)}/{(1/X)·(dS/dt)}=dX/dS=Yx/s  (1)

-   X: cell concentration(g-dry cell/L)-   S: carbon source concentration(g/L)

2. Exponential feeding method concerned with the C/N ratio: When thefeeding rate is linearly increased in the growth phase, q is higher thanthe critical point, for example, as described in Example 1 and Yx/s is0.200 to 0.500. On the other hand, when the feeding rate is linearlydecreased in the production phase, Yx/s is lower than 0.200. Moreover,the astaxanthin production is increased and the maximum production rateis obtained in this period. It is well known that the astaxanthinproduction by P. rhodozyma is typical non-growth associated typefermentation. The higher Yx/s is obtained in the growth phase, and thelower Yx/s is obtained in the production phase. Therefore, in order toenhance the cell growth extremely in the growth phase, which possesseshigher Yx/s, the exponential feeding method in the growth phase can beapplied.

Furthermore, in the production phase, in order to restrict the cellgrowth and efficiently convert glucose to astaxanthin, the glucoseconcentration can be controlled in the production phase.

When the exponential feeding in the growth phase is applied, μ can beset at 0.01 to 0.1 hour⁻¹. The feeding rate(F) in the growth phase isexpressed by following equation (2).F=(q·Vo·Xo·e ^(μt))/(S _(F) −S)  (2)

-   Vo: initial volume (L)-   Xo: initial cell concentration (g-dry cell/L)-   S_(F): carbon source concentration in feed medium (g/L)-   S: carbon source concentration in culture broth (g/L)

Moreover, the relationship between the amount of NH₄OH addition and theglucose consumption based on the result of the typical fed-batchfermentation method (the control method) is applied for the glucoseconcentration control in the culture broth. It means the pH-stat methodfor glucose concentration control based on the NH₄OH addition (formaintaining pH). In the production phase, the glucose concentration canbe controlled between 0 and 70 g/L, preferably between 0 and 10 g/L.

For instance, by using the 0 g/L glucose control, the final astaxanthinconcentration can be higher than that in the control method. The maximumastaxanthin production rate can be more than 30.0% higher than that ofthe control method. As described in Example 3, when the glucose 0 g/Lcontrol was used, the C/N ratio in the production phase was 1.08 timeshigher than the glucose 2.0 g/L control, because the NH₄OH addition inthe glucose 0 g/L control was smaller than that in the glucose 2.0 g/Lcontrol. It is known that the increment of the C/N ratio of feedingmedium in the production phase enhances the astaxanthin production 1.5times higher than the batch fermentation using the same amount ofglucose. Therefore, it is thought that the astaxanthin production usingthe glucose 0 g/L control is larger than that using the glucose 2.0 g/Lcontrol.

Moreover, using NaOH solution as a pH control reagent in the productionphase is the effective method for the astaxanthin production by theexponential feeding method with glucose 0 g/L control, because the C/Nratio in the production phase can be enhanced. The average astaxanthinproductivity by using NaOH solution as a pH control reagent in theproduction phase with an aim to increase the C/N ratio in the productionphase, can be more than 10.0% higher than by using NH₄OH solution. Theexponential feeding method with glucose 0 g/L control using NaOHsolution as a pH control reagent can be shortened more than 26.0% of thetotal fermentation period as compared to the control method. Thisexponential feeding method with glucose 0 g/L control can enhance theastaxanthin average productivity at least 13.0% higher than the controlmethod.

3. The modified exponential feeding method: In order to enhance thetotal astaxanthin production and utilize higher astaxanthin averageproductivity of the exponential feeding method, the exponential feedingmethod can be improved by increasing the feeding amount of glucose andexpanding the feeding period to the range of 10 to 50% longer. Effect ofthis expanding the feeding period method is ‘the modified exponentialfeeding method’ by enhancement of the feeding amount of glucose onastaxanthin fed-batch fermentation.

In the modified exponential feeding method, the exponential feeding isapplied in the growth phase till the middle stage of the fermentation.After that, the feeding rate of the production phase is kept constant atthe range of 10 to 100% of the maximum feeding rate in the growth phase.By using this modified exponential feeding method as described inExample 5, the final astaxanthin concentration was 5.8% higher than theconcentration by the control method, and the total astaxanthinproduction was at least 29.0% higher than that by the control method.The modified exponential feeding method can enhance the astaxanthincontent in dry cell more than 5.60% higher than the content by thecontrol method.

4. Effect of the glucose concentration in the culture broth on theastaxanthin production: In the modified exponential feeding method, theglucose is accumulated in the culture broth at the middle of thefermentation period. It is known that the accumulation of glucose inculture broth reduces the astaxanthin production, especially theastaxanthin yield against carbon sources (e.g. glucose, sucrose, etc.).It is estimated that the ethanol formulation is induced by the Crabtreeeffect and the astaxanthin yield is decreased. From the relationshipbetween the max glucose accumulation rate and the average specificastaxanthin production rate (p), p is gradually decreased to 71.0% ofthe average specific astaxanthin production rate at the no glucoseaccumulation phase, when the glucose begins to be accumulated in culturebroth.

The accumulation of glucose in the culture broth inhibits the ability toproduce astaxanthin per unit of cells. Therefore, for the purpose ofenhancing the astaxanthin yield, it is necessary to construct the newfeeding method for no glucose accumulating in the fermentation broth.Furthermore, in order to feed the glucose solution as large as possible,we have found the novel feeding method based on glucose consumption ratederived from the exponential feeding method.

5. The glucose consumption rate (GCR) feeding method: The GCR feedingmethod is based on the glucose consumption rate (GCR) in the fed-batchfermentation. The practical total glucose consumption profile can bederived from the exponential glucose feeding profile, e.g. as shown inExample 7.

Based on this feeding rate profile, the effect of the GCR feeding methodcan be investigated. In the GCR feeding method, the glucoseconcentration in the culture broth is able to keep around 0 g/L. Inspite of the same volume of the nutrient medium, the final totalastaxanthin production by using the GCR feeding method will be at least8.0% higher than that by using the exponential feeding method. Theaverage astaxanthin productivity by the GCR feeding method can be morethan 7.0% higher than that by the exponential feeding method.

These results show that the GCR feeding method is more effective thanthe exponential feeding method in order to enhance the astaxanthinproduction. In the GCR feeding method, the feeding amount of glucose canbe enhanced and the feeding period may be expanded.

6. The Max GCR feeding method: Furthermore, in order to enhance thefeeding amount of glucose without accumulating the glucose in thefermentation, the new GCR feeding method based on the maximum glucoseconsumption rate (the Max GCR feeding method) can be constructed byinvestigating the effects of the feeding profile that combined GCRfeeding and the constant feeding of keeping the maximum feeding rate inthe GCR feeding method in the production phase.

The glucose accumulation in the culture broth affects the astaxanthinyield, and the yield against glucose used is lower than the yield byusing the control method. If the new GCR feeding method is applied inorder not to accumulate glucose in the fermentation broth, it is thoughtthat the astaxanthin production yield can be remarkably enhanced. Inorder to investigate the Max GCR feeding method, the glucose consumptionrate based on the feeding method that combined the GCR feeding with theconstant feeding as described in Example 8 and the feeding rate profilebased on the glucose consumption rate was calculated. In Example 9 themax glucose feeding rate profile calculated from the feeding method thatcombined the GCR feeding with the constant feeding as described inExample 8 is shown.

The max glucose feeding rate profile can be set valuable depending oncompositions of the various nutrient media or fermentation conditions.Firstly, the Max GCR feeding method using NH₄OH and NaOH solution for pHcontrol can be applied based on the max glucose feeding rate profile.The total astaxanthin production by the Max GCR feeding method can beenhanced at least 4.0% higher than that by the control method. Themaximum astaxanthin production rate can be obtained more than 20% higherthan the rate by using the control method.

However, this method sometimes accumulates glucose in the culture broth.Therefore, in order to accumulate no glucose in the culture broth, theMax GCR feeding method using only NH₄OH solution for pH control can beapplied. By using this method, the glucose concentration in the culturebroth can maintain 0 g/L during the whole fermentation period. The totalastaxanthin production by this method can be enhanced more than 12%higher than that by the control method.

The astaxanthin production yield against glucose used by this Max GCRfeeding method can be increased by at least 4.0% higher than that by thecontrol method. The average astaxanthin production rate can be obtainedmore than 21% higher than the rate of the control method. Furthermore,this Max GCR feeding method using only NH₄OH solution for pH control canshorten the fermentation period (at least 11% of the fermentation periodcan be cut down in the comparison with the control method) and enhancethe astaxanthin productivity.

The present invention is illustrated by the following examples.

EXAMPLE 1 Typical Cultivation of P. rhodozyma ATCC96594 Mutant Strainfor the Astaxanthin Production

1. Seed culture preparation: P. rhodozyma ATCC96594 mutant strain wasused as seed strain. The Phaffia strain was propagated by beingtransferred from a slant to 500 ml Erlenmeyer flasks containing 50 ml ofYM medium in which the cells were cultured with shaking under sufficientaeration at 20 to 22° C. for 3 days. This seed culture was transferredto 500 ml Erlenmeyer flasks containing 100 ml of YM medium, andincubated for 2 days on a rotary shaker under sufficient aeration at 20to 22° C.

2. The fed-batch fermentation for astaxanthin production: 200 ml of theculture was transferred to a 5 L fermentor containing 1.75 L of thenutrient medium. The culture was subjected to batch growth at 20 to 22°C. until a yeast dry matter content of 1 g/L was obtained. Thereafter,the solution of sugar supply was started and the fed-batch fermentationwas performed at 20 to 22° C.

The nutrient medium had the following composition: 20 g/L of molasses,0.6 g/L of diammonium sulphate, 0.8 g/L of diammonium hydrogenphosphateand 0.125 g/L of magnesium sulphate which altogether were boiled up inthe fermentor for 30 min together with a suitable amount of water (1.75L in the 5 L propagation fermentor). The medium, which was fed to thefermentor in the fed-batch fermentation, contained 716.0 g/L of glucoseor sucrose. All the chemicals were of food grade. The molasses were beetsugar molasses from Midwest agriculture, US.

The fermentation was carried out in the glass jar fermentor, D-type(Able, Tokyo, Japan) with a total volume of 5 L with a top drive systemand temperature, pH, DO (dissolved oxygen) and exhaust gas monitor. Theinitial working volume and the feeding volume were 1.75 L and 2.0 L,respectively. The strains were propagated in fermentors at pH 5.5controlled with 12.5 w/v % NH₄OH solution and NaOH solution. Temperaturewas controlled at 20 to 22° C. and DO was controlled by agitation andairflow to between 10% and 90% saturation. During the growth phase, thefeeding medium containing glucose or sucrose fed to the main fermentorwith the aim of reaching the specific growth rate of the yeast cells ofμ: 0.04-0.05 h⁻¹. The feeding rate was gradually increased to so as notto accumulate the nutrient and ethanol in the culture broth.

Having achieved the desired yeast solids and prior to the accumulationof the nutrient source, the feed rate was reduced to about 50% of themaximum feed rate during the growth phase. During this period of reducednutrient source feed, namely the production phase, the yeast cellscontinued to produce astaxanthin, but the growth in the number of cellswas restricted. For example, in the production phase, the feeding ratewas linearly reduced to about 50% of the maximum feed rate for about 100hours.

EXAMPLE 2 Feeding Strategies Concerned with the Cell Growth Phase andthe Astaxanthin Production Phase

In the typical astaxanthin fed-batch fermentation described in example1, the specific glucose consumption rate (q) and the specific growthrate (μ) could be calculated and illustrated by a graph. When q and μwere 0.085 hours⁻¹ and 0.017 hour⁻¹, respectively, the slope of the linewas drastically changed and critical point, (q, μ)=(0.085, 0.017). Theslope μ/q means the cell yield against consumed glucose (Yx/s) as theequation (1) as described above.

When the feeding rate was linearly increased in the growth phase, μ washigher than 0.085 hour⁻¹ and Yx/s was 0.213 to 0.405. On the other hand,when the feeding rate was linearly decreased in the production phase,Yx/s was lower than 0.201. Moreover, the astaxanthin production wasincreased and the maximum production rate was obtained in this period.It is well known that the astaxanthin production by P. rhodozyma istypical non-growth associated type fermentation.

The higher Yx/s was obtained in the growth phase, and the lower Yx/s wasobtained in the production phase. Therefore, in order to enhance thecell growth extremely in the growth phase, which possessed higher Yx/s,and restrict the cell growth and efficiently convert glucose toastaxanthin in the production phase, the exponential feeding method inthe growth phase and the glucose concentration control in the productionphase were applied to the feeding strategies for the astaxanthinfed-batch fermentation.

EXAMPLE 3 Exponential Feeding Method Concerned with Effects of the C/NRatio on the Astaxanthin Fed-batch Fermentation

When the exponential feeding in the growth phase was applied, μ was setat 0.04 hour⁻¹. The feeding rate (F) in the growth phase is expressed bythe equation (2) as described above.

In this investigation, q, Vo, Xo, S_(F) and S were set at 0.08262hour⁻¹, 1.75 L, 9.92 g-dry cell/L, 716.0 g/L, and 0 g/L, respectively.Consequently, F in the growth phase is calculated as the followingequation (3),F=(0.08262·1.75·9.92·e ^(0.04t))/(716.0−0)=0.002003·e ^(0.04t)  (3)

Moreover, the relationship between the amount of NH₄OH addition and theglucose consumption based on the result of Example 1 was investigated.Two linear relationships (I and II) between the amount of NH₄OH additionand the glucose consumption were obtained and the critical point (NH₄OHaddition, Glucose consumption)=(3.95, 13.1) was introduced at 80 hoursfrom the beginning of the fermentation. In the growth phase, the slopeof the line was steep (I), in the production phase, the slope was gentle(II). Therefore, in order to control glucose 0 g/L in the productionphase, the medium feeding method based on the NH₄OH addition (formaintaining pH) coming from the slope II, namely, pH-stat method forglucose concentration control was applied. The glucose concentrationcontrols for the production phase were performed 0 and 2.0 g/L by 5 Ljar fermentation in the same manner as Example 1. Besides, the glucose2.0 g/L control was operated by the on-line glucose controller (theon-line biochemical controller BF-410: Able, Tokyo, Japan) in order toexplore the effects of the C/N ratio on the astaxanthin production fromthe beginning of the feeding.

These two conditions were compared with the typical feeding methoddescribed in Example 1 (the control method). Comparison of thefermentation activity between the three conditions is shown in Table 1.The final astaxanthin concentration in the 0 g/L glucose control was6.60% higher than that in the control method. The final astaxanthinconcentration in the 2.0 g/L glucose control was 13.5% smaller than thatin the control method.

The maximum astaxanthin production rate of the 0 g/L glucose control was32.9% higher than that of the control method.

When the glucose 0 g/L control was used, the C/N ratio in the productionphase was 1.08 times higher than the glucose 2.0 g/L control, becausethe NH₄OH addition in the glucose 0 g/L control was smaller than that inthe glucose 2.0 g/L control. By the increment of the C/N ratio offeeding medium in the production phase, the astaxanthin production usingthe glucose 0 g/L control was larger than that using the glucose 2.0 g/Lcontrol.

TABLE 1 Fermentation activity in the astaxanthin fed-batch fermentationusing a 5 L fermentor Typical feeding method Glucose 0 g/L Glucose 2.0g/L Status (Control) control control Final ASTA conc. 100 106.6 86.5 MaxASTA 100 132.9 85.5 production rate ASTA: astaxanthin; the data areexpressed as relative values

EXAMPLE 4 New Feeding Method of Exponential Feeding and GlucoseConcentration Control for the Astaxanthin Fed-batch Fermentation

Moreover, the effects of C/N ratio on the exponential feeding methodwith glucose 0 g/L control were investigated by using NaOH solution as apH control reagent in 5 L jar fermentations. The fermentations wereperformed in the same manner as in Example 1. In the case of theexponential feeding method, the glucose 0 g/L control in the productionphase was carried out by the feeding method that the feeding rate islinearly decreased from 22.61 to 17.63 g-feed solution/hour. The averageastaxanthin productivity by using NaOH solution as a pH control inproduction phase with an aim to increase the C/N ratio in the productionphase, was 13.5% higher than by using NH₄OH solution.

Table 2 shows the comparison between the typical feeding methoddescribed in Example 1 (the control method) and the exponential feedingmethod by using NaOH solution as a pH control in the production phase inthe astaxanthin production by fed-batch mode. The total fermentationperiod in the exponential feeding method was 41 hours shorter than thatin the control method. The astaxanthin concentration by the exponentialfeeding method was 5.7% higher than that by the control method at 175hours from the beginning of the fermentation. The astaxanthin averageproductivity in the exponential feeding method was 13.9% higher thanthat in the control method.

TABLE 2 Effect of the exponential feeding method by using NaOH solutionas a pH control in the production phase on the astaxanthin production bythe fed-batch mode Exponential feeding Typical method by using NaOHfeeding method solution for the Status (Control) pH control Final ASTAconc. at 175 th h 100 105.7 average ASTA production rate 100 113.9 ASTA:astaxanthin; the data are expressed as relative values

EXAMPLE 5 Feeding Method of the Modified Exponential Feeding for theAstaxanthin Fed-batch Fermentation

In order to enhance the total astaxanthin production and utilize higherastaxanthin average productivity of the exponential feeding method, theexponential feeding method was improved by increasing the feeding amountof glucose and expanding the feeding period from 117 hours to 156 hours.The effect of this expanding the feeding period method, in other words,‘the modified exponential feeding method’ by enhancement of the feedingamount of glucose on astaxanthin fed-batch fermentation wasinvestigated. In the modified exponential feeding method, the feedingrate of the production phase was kept at 19.7 g/hr till 186.5 hours fromthe beginning of the fermentation (156 hours from the beginning of theglucose feeding).

Table 3 shows the comparison between the typical feeding methoddescribed in Example 1 (the control method) and the modified exponentialfeeding method.

The final astaxanthin concentration by the modified exponential feedingmethod was 5.8% higher than that by the control method. The totalastaxanthin production by the modified exponential feeding method was29.3% higher than that by the control method. Although the glucose wasaccumulated in the culture broth from 98 hours to 192 hours by using themodified exponential feeding method, the fed glucose was completelyconsumed at the end of the fermentation. The astaxanthin content in drycells in the modified exponential feeding method was 5.60% higher thanthat in the control method.

TABLE 3 Effect of the modified exponential feeding method on theastaxanthin pro- duction by the fed-batch mode Typical feeding methodModified exponential Status (Control) feeding method Final ASTA conc.100 105.8 Total amount of ASTA 100 129.3 ASTA content in dry cell 100105.6 ASTA: astaxanthin; the data are expressed as relative values

EXAMPLE 6 Effect of the Glucose Concentration in the Culture Broth onthe Astaxanthin Fed-batch Fermentation

As described in Example 5, the glucose was accumulated in the culturebroth by using the exponential feeding method. The total astaxanthinproduction and yield were lower than the production by the controlmethod described in Example 1. As the ethanol formulation may be inducedby the Crabtree effect, the astaxanthin yield was decreased.Accordingly, the relationship between the max glucose accumulation rateand the average specific astaxanthin production rate (p) wasinvestigated.

When the glucose began to be accumulated in culture broth, ρ wasgradually decreased to 71.0% of the average specific astaxanthinproduction rate at the no glucose accumulation phase. These results showthat the accumulation of glucose in culture broth inhibited the abilityto produce astaxanthin per unit of cells. It was estimated that theethanol formation was induced by the Crabtree effect and the astaxanthinyield was decreased.

EXAMPLE 7 Astaxanthin Fed-batch Fermentation by the New Feeding MethodBased on the Practical Glucose Consumption Rate (The GCR Feeding Method)

For the purpose of enhancing the astaxanthin yield, it is necessary toconstruct the new feeding method for no glucose accumulating in thefermentation broth. Furthermore, in order to feed the glucose solutionas large as possible, we studied the feeding method based on glucoseconsumption rate derived from the exponential feeding method.

The practical total glucose consumption profile differed from theexponential glucose feeding profile. Based on this result, the practicalglucose consumption rate profile was calculated.

The practical profile of glucose consumption rate was expressed as acubic equation by the multiple regression analysis. From the glucoseconsumption rate profile, a practical feeding rate profile showingglucose feeding pattern based on glucose consumption was established.

Based on this feeding rate profile, the effect of GCR feeding method wasinvestigated. Table 4 shows the comparison between the exponentialfeeding method and the GCR feeding method. In the GCR feeding method,the glucose concentration in the culture broth was maintained around 0g/L. In spite of the same condition that the initial volume was 1.75 Land the feeding volume was 2.00 L, the final total astaxanthinproduction by using the GCR feeding method was 8.99% higher than that byusing the exponential feeding method. Furthermore, the fermentationperiod by the GCR feeding method was able to set 168 hour, which was thesame period by the exponential feeding method. Then, the averageastaxanthin productivity by GCR feeding method was 7.60% higher thanthat by the exponential feeding method.

TABLE 4 The effect of the modified exponential feeding method on theastaxanthin production by fed-batch mode Modified exponential Statusfeeding method GCR feeding method Final ASTA conc. 100 107.6 Totalamount of ASTA 100 109.0 Average ASTA productivity 100 107.6 ASTA:astaxanthin; the data were expressed as relative values

EXAMPLE 8 Enhancement of the Feeding Amount of Glucose in theAstaxanthin Fed-batch Fermentation by the Modified GCR Feeding Method

In the GCR feeding method, the feeding amount of glucose was enhancedand the feeding period was expanded to 156 hours. Table 5 shows theeffects of the modified GCR feeding methods on astaxanthin production byfed-batch fermentation.

The modified GCR feeding method means the method that combines the GCRfeeding method till 111 hours from the beginning of the glucose feedingand the feeding method of a linear decrement of the control method after111 hours as described in Example 1.

The final total astaxanthin production by the modified GCR feedingmethod was 5.5% higher than that by the control method. Furthermore, theeffects of the feeding profile that combined GCR feeding and theconstant feeding of keeping the maximum feeding rate in the GCR feedingmethod, which was reached after 66th hour from the beginning of theglucose feeding. Namely, the GCR feeding method was used from thebeginning of the glucose feeding to 66 hours, and the constant feedingwhich was kept 30.4 g-feed solution 1 hour of the max. feeding rate ofthe GCR feeding method, was used from 66 hours to 156 hours.

Table 5 also shows the results of the effects of the feeding method thatcombined the GCR feeding with the constant feeding. The final totalastaxanthin production by this feeding method was 19.8% higher than thatby the control method and about 14% higher than that by the modified GCRfeeding method.

TABLE 5 The effects of the modified GCR feeding methods on astaxanthinproduction by fed-batch fermentation Modified Typical feeding ModifiedGCR GCR + constant Status method (Control) feeding method feeding methodTotal amount 100 105.5 119.8 of ASTA ASTA: astaxanthin; the data wereexpressed as relative values

EXAMPLE 9 Astaxanthin Fed-batch Fermentation by the Max GCR FeedingMethod

In Example 8 glucose in culture broth begun to accumulate gradually from120 hours and the glucose concentration reached to 81.7 g/L at 192ndhour from the beginning of the fermentation. Finally, the glucoseconcentration was 33.6 g/L at the end of the fermentation. The glucoseaccumulation in culture broth affected the astaxanthin yield. Therefore,the new GCR feeding method based on the maximum glucose consumption rate(the Max GCR feeding method) was constructed. In order to investigatethe Max GCR feeding method, the glucose consumption rate based on thefeeding method that combined the GCR feeding with the constant feedingas described in Example 8 and the feeding rate profile based on theglucose consumption rate was calculated.

The max glucose feeding rate profile was calculated from the feedingmethod that combined the GCR feeding with the constant feeding asdescribed in Example 8.

The maximum feeding rate calculated from the practical glucoseconsumption rate was 28.7 g/hr at 66 hours from the beginning of theglucose feeding. After 66 hours because of the decrement of the glucoseconsumption rate, the feeding rate had to be set to less than 19.0 g/hrin order to keep the glucose concentration around 0 g/L. Therefore,based on the max glucose feeding rate profile, Max GCR feeding methodusing NH₄OH and NaOH solution for pH control was applied. The initialvolume and the feeding volume were set at 1.50 L and 2.25 L,respectively.

Table 6 shows the effect of the Max GCR feeding method using NH₄OH andNaOH solution for pH control on astaxanthin production by fed-batchfermentation. At 216 hours from the beginning of the fermentation, thetotal astaxanthin production by the Max GCR feeding method was 4.21%higher than that of the control method.

Furthermore, in the Max GCR feeding method, the maximum astaxanthinproduction rate calculated from 95 hours to 122 hours was and 20.1%higher than the rate by using the control method. However, glucose inthe culture broth of the Max GCR feeding method using NH₄OH and NaOHsolution for pH control was accumulated 19.9 g/L at 175 hours from thebeginning of the fermentation.

The astaxanthin production yield against glucose used by the Max GCRfeeding method was lower than that by the control method, since theglucose was accumulated in the culture broth.

Next, in order to accumulate no glucose in the culture broth, the MaxGCR feeding method using only NH₄OH solution for pH control wasestablished.

Table 7 shows the effect of Max GCR feeding method using only NH₄OHsolution for pH control on Astaxanthin production by fed-batchfermentation. In Table 7, the Max GCR feeding method using only NH₄OHsolution could maintain 0 g/L of the glucose concentration in theculture broth during the whole fermentation period. At 216 hours fromthe beginning of the fermentation, the total astaxanthin production bythis method was 12.1% higher than that by the control method.

The astaxanthin production yield against glucose used by this Max GCRfeeding method was 4.7% higher than that of the control method. In thisMax GCR feeding method, the average astaxanthin production rate was21.9% higher than the rate of the control method. From the totalastaxanthin production profile, this Max GCR feeding method using onlyNH₄OH solution for pH control in both the growth phase and theastaxanthin production phase could shorten the fermentation period(11.2% of the fermentation period was cut down in the comparison withthe control method) and enhance the astaxanthin productivity.

TABLE 6 Effect of the Max GCR feeding method using NH₄OH and NaOHsolution for pH control on astaxanthin production by fed-batchfermentation Max GCR feeding method by using Typical feeding NaOH andNH₄OH Status method (Control) for pH control Total amount of ASTA 100104.2 Max ASTA production rate 100 120.1 Max glucose accumulation inculture broth (g/L)  0  19.9 ASTA: astaxanthin; the data were expressedas relative values

TABLE 7 Effect of the Max GCR feeding method using NH₄OH and NaOHsolution for pH control on astaxanthin productioii by fed-batchfermentations Max GCR feeding Typical method by using feeding methodNH₄OH solution Status (Control) for pH control Total amount of ASTA 100112.1 Max glucose accumulation in  0  0   culture broth (g/L) ASTAproduction yield 100 104.7 Final ASTA conc. 100 108.3 Average ASTAproductivity 100 121.9 ASTA: astaxanthin; the data were expressed asrelative values

1. A fermentation method of astaxanthin using Phaffia rhodozymacomprising the steps of: (a) in the growing phase, feeding of a nutrientmedium containing glucose or sucrose based on the specific growth rate(μ) of Phaffia rhodozyma cells, and (b) in the astaxanthin productionphase, feeding of the nutrient medium based on the astaxanthinproduction rate, while keeping the glucose concentration in thefermentation broth at 0 g/L during the whole fermentation period.
 2. Thefermentation method according to claim 1, wherein the fermentation iscarried out at a μ range between 0.01 and 0.10 h⁻¹.
 3. The fermentationmethod according to claim 1, wherein the pH control reagent is NH₄OHsolution, NaOH solution or both.
 4. The fermentation method according toclaim 1, wherein the fermentation is carried out at a pH between 4.5 and7.0.
 5. The fermentation method according to claim 1, wherein thefermentation is carried out at a temperature in the range of from 15 to24° C.
 6. The method according to claim 1, wherein the fermentation iscarried out at DO between 10 and 90%.
 7. The fermentation methodaccording to claim 1, wherein the nutrient medium has at least onecarbon energy source selected from the group consisting of polymerizedforms of glucose, sucrose and other polysaccharides, molasses and cornsyrup, glycerol and other polyols, and carboxylic acids, and, at leastone nitrogen source selected from the group consisting of yeast extract,meat-extract, peptone, casein, corn steep liquor, urea, amino acid,nitrates, and ammonium salts.
 8. The fermentation method according toclaim 1, wherein the concentration of D-glucose or sucrose in thenutrient medium is from about 10 g/L to about 800 g/L.
 9. Thefermentation method according to claim 1, wherein the fermentation iscarried out at gassing rates of about 0.01 to about 2.0 volume ofgas/volume of medium/min. in the fermentation vessel.
 10. Thefermentation method according to claim 1, wherein Phaffia rhodozyma isPhaffia rhodozyma ATCC96594.